Applicants"" invention is directed to the field of evaporative light scattering detection and methods.
Evaporative light scattering detection is a method of detecting samples that have been previously separated in various chromatography methods such as, for example, High Performance Liquid Chromatography (HPLC), Gel-Permeation Chromatography (GPC), High Performance Centrifugal Partition Chromatography (HPCPC), Field Flow Fractionation (FFF), and Supercritical Fluid Chromatography (SFC). Evaporative light scattering detection is preferably used when the sample components (e.g., components to be detected) have lower volatility than the mobile phase. A wide variety of sample types can be detected in evaporative light scattering detection. Such sample types include, for example, lipids, triglycerides, surfactants, polymers, underivatized fatty and amino acids, carbohydrates and pharmaceuticals.
Generally, evaporative light scattering detection involves four main steps: 1) nebulization of the chromatography effluent, (which consists of the mobile phase and the sample), into an aerosol of particles, 2) evaporation of the mobile phase, 3) light scattering by the sample particles, and 4) detection of the scattered light. There are two principal types of devices used in evaporative light scattering detection known in the art. In the first type (the xe2x80x9csingle flowxe2x80x9d design), the nebulized chromatography effluent is immediately introduced into a heated drift tube where the mobile phase is evaporated. The sample particles are then flowed from the heated drift tube to an optical cell where light scattering and detection occurs. One such example of this type of device (the Alltech Model 500 ELSD) is sold by the assignee of this application, ALLTECH ASSOCIATES, INC. Details concerning the design and operating parameters for such a device are disclosed in the Operating Manual for the Alltech Model 500 ELSD, which is incorporated herein by reference.
In the second type of device, (the xe2x80x9csplit-flowxe2x80x9d design), the nebulized chromatography effluent is first flowed through a nebulization chamber before entering the heated drift tube. In the nebulization chamber, the nebulized chromatography effluent is split, namely, the larger droplets are eliminated by condensation/impaction on the walls of the nebulization chamber. This condensate is drained to waste. Only the smaller nebulized droplets are subsequently flowed to the drift tube where the mobile phase (which is now free of the larger droplets) is more easily evaporated. Thereafter, the sample particles are flowed to the optical cell for light scattering and detection. Devices of this design type are available from, for example, SEDERE or EUROPSEP INSTRUMENTS.
The above-described design types have particular advantages depending on the mobile phase and the sample type. The single flow design is preferred for use in applications involving relatively non-volatile sample types and volatile organic mobile phases. Because all of the sample enters the optical cell in this design, response and sensitivity is maximized.
However, the split-flow design is preferably used with highly aqueous mobile phases and semi-volatile sample types. Highly aqueous mobile phases generally require higher evaporation temperatures. If the sample is volatile at these higher evaporation temperatures, sample loss is incurred during the evaporation step resulting in poorer sensitivity. By using the split-flow design, the evaporation temperature is reduced. This is accomplished by removing the larger mobile phase droplets in the nebulized chromatography effluent before the evaporation step. By removing the larger droplets, a smaller and more uniform particle size distribution is achieved in the mobile phase, which leads to lower evaporation temperatures. The lower evaporation temperatures, in turn, lead to less sample loss during the evaporation step. However, for non-volatile sample types and organic mobile phases, the split-flow design is generally less preferred because some of the non-volatile sample may be lost during the splitting of the chromatography effluent.
Another problem with devices of the split-flow design is that the split ratio of the sample (i.e., the amount that goes to waste versus the amount that is ultimately detected) is affected by, among other things, the laboratory temperature. In other words, fluctuations in laboratory temperatures lead to fluctuations in droplet size in the nebulized chromatography effluent. Thus, as ambient and/or laboratory temperatures fluctuate, the split ratio and corresponding reproducibility of sample detection may vary from run to run.
As is evident from the above-discussion, depending on the mobile phase and the sample type being detected, one evaporative light scattering detection design and method is advantageous over the other. However, laboratories often work with both aqueous and organic mobile phases and various sample types with different volatilities. Ideally, laboratories would have available both design types for evaporative light scattering detection. However, in order to have this benefit, the laboratory would need to purchase two separate devices, which can be expensive. It would be advantageous and constitute an improvement in the art if an evaporative light scattering detection device and system were developed which could be quickly and inexpensively converted between the single flow and split flow designs. Applicants have developed such a device and system. Moreover, with respect to the split-flow design, Applicants invention addresses the problem of the variation in split ratio caused by fluctuating laboratory temperatures.
In one respect, the present disclosure is directed to a system for evaporative light scattering detection which allows for quick and easy conversion between a single flow design and a split flow design, depending on the mobile phase and sample types to be detected. The system includes an evaporative light scattering detection device comprising a removably attached nebulizer in fluid communication with a heated drift tube, a light source, and a detector for detecting scattered light. The system also includes a low temperature adaptor comprising a nebulization chamber and a coil. The system further includes a connection tube for providing a fluid connection between the light scattering detection device and the low temperature adaptor for converting from a single flow to the split flow designs. One end of the connection tube is attached to the low temperature adaptor and the other end of the connection tube is removably attached to the evaporative light scattering detection device such that the connection tube provides fluid communication between the low temperature adaptor and the evaporative light scattering device. The low temperature adaptor is connected to the evaporative light scattering device by first removing the nebulizer from the detection device and attaching in its place the connection tube to provide fluid communication between the low temperature adaptor and the detection device. The low temperature adaptor further comprises a nebulizer. The nebulizer for the low temperature adaptor may be the nebulizer removed from the evaporative light scattering device or a second nebulizer.
The low temperature adapter in the above system further preferably comprises a sweep gas channel for introducing into the nebulization chamber sweep gas independently of the nebulizing gas. The sweep gas is for assisting in the evaporation of the mobile phase. Also, heat tape is preferably affixed to the nebulization chamber and the coil of the low temperature adaptor in the above system at pre-determined intervals for controlling the temperature of the nebulization chamber and coil.
In another respect, the disclosure is directed to a low temperature adaptor for a light scattering detection device which reduces the temperature required to evaporate the mobile phase. The low temperature adaptor is especially preferred for aqueous mobile phases and semi-volatile sample types. The low temperature adaptor comprises a nebulization chamber, a coil and a connection tube for removably attaching the low temperature adaptor to the evaporative light scattering detection device such that the connection tube provides a fluid connection between the low temperature adaptor and the evaporative light scattering detection device. Heat tape is preferably affixed to the nebulization chamber and the coil at pre-determined intervals for controlling the temperature of the nebulization chamber and coil. The low temperature adaptor preferably further includes a sweep gas channel for introducing sweep gas into the nebulization chamber independently of nebulizing gas. Finally, the low temperature adapter further includes a nebulizer. The nebulizer may be the nebulizer removed from the evaporative light scattering detection device prior to connecting the low temperature adapter or a second nebulizer.
In another aspect, the disclosure concerns a method of evaporative light scattering detection which is substantially resistant to fluctuations in ambient temperature conditions. By substantially resistant to fluctuations in ambient temperature conditions, it is meant that the detection device of this invention provides consistent detection when laboratory temperatures fluctuate of from about 15xc2x0 C. to about 40xc2x0 C. The method comprises flowing nebulized chromatography effluent comprising mobile phase and sample to be detected through a nebulization chamber, wherein the temperature of the nebulization chamber is controlled by a heat source; reducing the particle size distribution of the nebulized chromatography effluent in the nebulization chamber; evaporating the mobile phase; and detecting the sample by evaporative light scattering detection. Preferably, the temperature in the nebulization is controlled by heat tape affixed to the nebulization chamber at predetermined intervals.